The long term goal is to understand in molecular terms, how a neurotropic herpesvirus invades and spreads inn the mammalian nervous system. Pseudorabies virus (PRV) is a well studied alpha herpesvirus causing a disease of economic impact in swine. PRV displays a striking neurotropism infecting both the peripheral and central nervous system of a variety of mammals not unlike the human alpha herpesviruses herpes simplex (HSV) or varicella zoster virus (VZV). Recently, we have shown that PRV mutants lacking two "non-essential" membrane proteins (gE and gI) display a distinct neurotropism phenotype in rats. After infection of the retina, mutants lacking gE or gI retain the ability to infect the circadian rhythm centers of the brain but have lost the ability to infect the visual centers. This inability to infect a functionally distinct subset of neurons is accompanied by a reduction in virulence (mean time to symptoms or death). These observations have significance not only for detailed analysis of the molecular mechanisms of viral pathogenesis, but also for studying structure and function in the nervous system. Our approach centers on a genetic analysis of the PRV gE and gI genes. In specific aim 1, mutants will be constructed based on the hypothesis that specific neurotropism and virulence reflect distinct and separate gE/gI interactions with the host. By isolating defined gE/gI mutants and screening them for defects in specific neurotropism or virulence, we can determine if these two phenotypes can be resolved. In specific aim 2, since all alpha herpesviruses have homologs of gE and gI, we should as a general principle, be able to gain insight into common structure and function by measuring complementation for a given defect after exchange of homologs or by making hybrid proteins. We propose to test this idea by determining if the gE/gI homologs of VZV can complement either or both of the novel phenotypes of PRV gE/gI mutants. Finally in specific aim 3, we will develop an in vivo genetic selection for novel PRV mutants based on inability of gE and gI mutants to be transported from the rat retina to the visual centers of the brain. A clear genetic definition of functional domains in the gE and gI proteins will give significant insight into molecular mechanisms of tropism and pathogenesis.